Journal: Nucleic Acids Research

Article Title: A 96-well DNase I footprinting screen for drug–DNA interactions

doi: 10.1093/nar/gkm467

Figure Lengend Snippet: Infrared gel electrophoresis images of DNase I footprinting products following incubation of compounds 1–6 with TOPOIIα promoter DNA. Compounds were incubated for 17 h with TOPOIIα promoter DNA IR-labelled on the sense (A) or antisense (B) strand. Each gel consists of 50 lanes: two GA marker lanes (GA) and six sets of eight lanes for compound (l–r; 0, 0.0016, 0.008, 0.04, 0.2, 1, 5 and 25 μM). The sequence positions relating to Supplementary Figure 1 are given vertically.

Article Snippet: The proximal 705 bp of the human Topoisomerase II alpha (TOPOIIα) promoter was amplified by PCR from human genomic DNA (Promega) using primers TIIA-F (CACCGCACACAGCCTAC) and TIIA-R (TGGTGACGGTCGTGAAG) (Supplementary Figure 1).

Techniques: Nucleic Acid Electrophoresis, Footprinting, Incubation, Marker, Sequencing

Journal: Nucleic Acids Research

Article Title: A 96-well DNase I footprinting screen for drug–DNA interactions

doi: 10.1093/nar/gkm467

Figure Lengend Snippet: A comparison of footprint profiles of compounds 1–6 on the proximal region of the TOPOIIα promoter. Single footprint profiles were program-generated starting with the IR footprint gel image shown in A. Where no footprinting is seen, the sequence has been given an arbitrary score of −3.9 (log 125 μM). The DNA sequence is given in Supplementary Figure 1.

Article Snippet: The proximal 705 bp of the human Topoisomerase II alpha (TOPOIIα) promoter was amplified by PCR from human genomic DNA (Promega) using primers TIIA-F (CACCGCACACAGCCTAC) and TIIA-R (TGGTGACGGTCGTGAAG) (Supplementary Figure 1).

Techniques: Generated, Footprinting, Sequencing

Journal: Developmental cell

Article Title: Hematopoietic Reprogramming in vitro Informs in vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells

doi: 10.1016/j.devcel.2016.02.011

Figure Lengend Snippet: E10.5 (left panels) and E12.5 (right panels) placentas were dissociated to a single cell suspension and analyzed. Prom1+Sca1+CD34+ cells are displayed for (A) FCS/SSC, CD49f and (B) CD31, CD105, VE-Cadherin, Tie2, CD41 and cKit expression. Red boxes highlight the phenotype Prom1+Sca1+CD45− cells.

Article Snippet: Ao, Aorta. ( D ) Fluidigm qRT-PCR analysis of Prom1 , Ly6a and CD45 in E10.5 AGM+VU endothelial (EC: CD144+(VE-Cadherin)Ter119−CD45−CD41−23GFP−), hemogenic endothelium (HE: CD144+Ter119−CD45−CD41−23GFP+) and HSPCs (CD144+Ter119−CD45−CD41+23GFP+) (mean ± SD, n=3).

Techniques: Expressing

Journal: Developmental cell

Article Title: Hematopoietic Reprogramming in vitro Informs in vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells

doi: 10.1016/j.devcel.2016.02.011

Figure Lengend Snippet: Placentas from (A) E10.5, (B) E11.5 and (C) E12.5 were stained with antibodies against CD133 (Prom1, green). The left panels show composite low magnification pictures of transversal sections of placentas. The region of the labyrinth that contains Prom1+ cells is highlighted (white circles).

Article Snippet: Ao, Aorta. ( D ) Fluidigm qRT-PCR analysis of Prom1 , Ly6a and CD45 in E10.5 AGM+VU endothelial (EC: CD144+(VE-Cadherin)Ter119−CD45−CD41−23GFP−), hemogenic endothelium (HE: CD144+Ter119−CD45−CD41−23GFP+) and HSPCs (CD144+Ter119−CD45−CD41+23GFP+) (mean ± SD, n=3).

Techniques: Staining

Journal: Developmental cell

Article Title: Hematopoietic Reprogramming in vitro Informs in vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells

doi: 10.1016/j.devcel.2016.02.011

Figure Lengend Snippet: (A) Hemogenic PS34 cells from E10.5 and E12.5 placentas were fractioned into CD45−cKit−, CD45−cKit+ and CD45+cKit+ populations. HSPCs and mature blood cells (MBC) were also sorted for comparison.

Article Snippet: Ao, Aorta. ( D ) Fluidigm qRT-PCR analysis of Prom1 , Ly6a and CD45 in E10.5 AGM+VU endothelial (EC: CD144+(VE-Cadherin)Ter119−CD45−CD41−23GFP−), hemogenic endothelium (HE: CD144+Ter119−CD45−CD41−23GFP+) and HSPCs (CD144+Ter119−CD45−CD41+23GFP+) (mean ± SD, n=3).

Techniques:

Journal: Developmental cell

Article Title: Hematopoietic Reprogramming in vitro Informs in vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells

doi: 10.1016/j.devcel.2016.02.011

Figure Lengend Snippet: (A) E10.5 AGM+VU regions were isolated, dissociated to a single cell suspension and analyzed by flow cytometry. PS34CD45− and PS34CD45+ cells are gated and displayed for the expression of the 23GFP Runx1 reporter. Percentages are shown as mean ± SD, n=3.

Article Snippet: Ao, Aorta. ( D ) Fluidigm qRT-PCR analysis of Prom1 , Ly6a and CD45 in E10.5 AGM+VU endothelial (EC: CD144+(VE-Cadherin)Ter119−CD45−CD41−23GFP−), hemogenic endothelium (HE: CD144+Ter119−CD45−CD41−23GFP+) and HSPCs (CD144+Ter119−CD45−CD41+23GFP+) (mean ± SD, n=3).

Techniques: Isolation, Flow Cytometry, Expressing

Journal: Developmental cell

Article Title: Hematopoietic Reprogramming in vitro Informs in vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells

doi: 10.1016/j.devcel.2016.02.011

Figure Lengend Snippet: (A) E12.5 PS34CD45− cells were co-cultured on OP-9 in the presence of cytokines (SCF, Fltl3L, IL-3, IL-6 and TPO) for 4 days. FACS analysis shows the generation of CD45+CD34+cKit+ cells that were tested for clonogenic activity.

Article Snippet: Ao, Aorta. ( D ) Fluidigm qRT-PCR analysis of Prom1 , Ly6a and CD45 in E10.5 AGM+VU endothelial (EC: CD144+(VE-Cadherin)Ter119−CD45−CD41−23GFP−), hemogenic endothelium (HE: CD144+Ter119−CD45−CD41−23GFP+) and HSPCs (CD144+Ter119−CD45−CD41+23GFP+) (mean ± SD, n=3).

Techniques: Cell Culture, Activity Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells regulate type 2 innate lymphoid cells via regulatory T cells through ICOS-ICOSL interaction.

doi: 10.1002/stem.3369

Figure Lengend Snippet: FIGURE 1 iPSC-MSCs inhibited ILC2 function in PBMCs from patients with allergic rhinitis. PBMCs isolated from AR patients and healthy subjects were cocultured with iPSC-MSCs. A, IL-13, IL-9, and IL-5 levels in the supernatant were analyzed by ELISA (n = 6 for HC, n = 8 for AR patients); B, IL-13, IL-9, and IL-5 mRNA levels in PBMCs were analyzed by qPCR (n = 6 for HC patients, n = 8 for AR patients). C, The intracellular IL-13+ and IL-9+ Lin−CRTH2+ cell levels in PBMCs were analyzed by flow cytometry (n = 8). Data are shown as mean ± SEM. *P < .05, **P < .01, and ***P < .001. AR, allergic rhinitis; HC, healthy control; ILC2, group 2 innate lymphoid cell; iPSC-MSCs, induced pluripotent stem cell-derived mesenchymal stem cells; Lin−, lineage negative; PBMCs, peripheral blood mononuclear cells

Article Snippet: Furthermore, ILC2s were isolated from Lin− cells using ILC2 Isolation Kit, human (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Control, Derivative Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells regulate type 2 innate lymphoid cells via regulatory T cells through ICOS-ICOSL interaction.

doi: 10.1002/stem.3369

Figure Lengend Snippet: FIGURE 2 MSCs directly enhanced the levels and functions of purified ILC2s. Lin−cells (A-C) or ILC2s (D) isolated from the buffy coat were cocultured with iPSC-MSCs. A, The intracellular IL-13+ and IL-9+ Lin−CRTH2+ cell levels were analyzed by flow cytometry (n = 6). B, IL-13, IL-9, and IL-5 levels in the supernatant were analyzed by ELISA (n = 6). C, The CRTH2+ Lin−cell and CD127+ Lin−cell levels were analyzed by flow cytometry (n = 6). Furthermore, sorted ILC2s were cocultured with iPSC-MSCs, IL-13, IL-9, and IL-5 levels (D, n = 6) in the supernatant were analyzed by ELISA. Data are shown as mean ± SEM. *P < .05, **P < .01, and ***P < .001. AR, allergic rhinitis; ILC2, group 2 innate lymphoid cell; iPSC-MSCs, induced pluripotent stem cell-derived mesenchymal stem cells; Lin−, lineage negative

Article Snippet: Furthermore, ILC2s were isolated from Lin− cells using ILC2 Isolation Kit, human (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Purification, Isolation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells regulate type 2 innate lymphoid cells via regulatory T cells through ICOS-ICOSL interaction.

doi: 10.1002/stem.3369

Figure Lengend Snippet: FIGURE 3 iPSC-MSCs enhanced ILC2 levels and function via direct cell-cell contact. Transwell culture system was employed to separate Lin−

Article Snippet: Furthermore, ILC2s were isolated from Lin− cells using ILC2 Isolation Kit, human (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques:

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells regulate type 2 innate lymphoid cells via regulatory T cells through ICOS-ICOSL interaction.

doi: 10.1002/stem.3369

Figure Lengend Snippet: FIGURE 4 iPSC-MSCs suppressed ILC2 function via Treg cells through ICOS-ICOSL interaction. nTreg cells or MSC-Treg were cocultured together with Lin−cells and/or iPSC-MSCs. IL-13 and IL-9 levels in the supernatant were analyzed by ELISA (A and B, n = 6). ICOS and ICOSL expression on Lin−cells (C), ICOS expression on Treg cells (D), and ICOSL expression on iPSC-MSCs (E) were analyzed by flow cytometry prior to and after coculture (n = 6). After Treg cells were pretreated with anti-ICOS mAb, intracellular IL-13+ Lin−CRTH2+ cell and IL-9+ Lin−CRTH2+ cell levels (F, n = 6), CRTH2+ Lin−cell levels (G, n = 6) were analyzed by flow cytometry. IL-13, IL-9, and IL-5 levels (H, n = 6) in the supernatant were analyzed by ELISA. Data are shown as mean ± SEM. *P < .05, **P < .01, and ***P < .001. ILC2, group 2 innate lymphoid cell; iPSC-MSCs, induced pluripotent stem cell-derived mesenchymal stem cells; Lin−, lineage negative; MSC-Treg, Treg cells were pretreated with iPSC-MSCs; nTreg, natural Treg cells; Treg, regulatory T cells

Article Snippet: Furthermore, ILC2s were isolated from Lin− cells using ILC2 Isolation Kit, human (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Derivative Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells regulate type 2 innate lymphoid cells via regulatory T cells through ICOS-ICOSL interaction.

doi: 10.1002/stem.3369

Figure Lengend Snippet: FIGURE 5 iPSC-MSCs inhibited ILC2 functions via IL-10 secreted by Treg cells. A, The production of IL-10 by Treg cells following interaction with iPSC-MSCs and Lin−cells were analyzed by ELISA (n = 6). An anti-IL-10 mAb was added into the coculture system to block IL-10 function. Intracellular IL-13+ and IL-9+ Lin−CRTH2+ cell levels (B), CRTH2+ Lin−cell levels (C) were analyzed by flow cytometry (n = 6). D, IL-13, IL-9, and IL-5 levels in the supernatant were analyzed by ELISA (n = 6). Data are shown as mean ± SEM. *P < .05, **P < .01, and ***P < .001. iPSC-MSCs, induced pluripotent stem cell-derived mesenchymal stem cells; Lin−, lineage negative; nTreg, natural Treg cells; Treg, regulatory T cells

Article Snippet: Furthermore, ILC2s were isolated from Lin− cells using ILC2 Isolation Kit, human (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Flow Cytometry, Derivative Assay